Background Toll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues. Methods TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed. Results In situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice. Conclusion TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7’s neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7’s primary role in neuronal tissues is not related to antiviral immunity.
Background: Asthma is characterized by excessive bronchoconstriction and cough. Airway nerves control these reflexes. In asthma, an abundance of airway eosinophils cause airway nerve dysfunction by altering neurotransmitter content and neuronal receptor expression. Objective: To characterize the effects of eosinophils on Transient Receptor Potential V1 (TRPV1) and substance P (SP) expression in dorsal root ganglia sensory neurons. Methods: Dorsal root ganglia were isolated from wild-type C57BL/6 mice, IL-5 transgenic mice with airway eosinophilia driven by high IL-5 (IL5tg, NJ.1726 lineage), and transgenic eosinophildeficient (PHIL) mice. Ganglia were immersed for 24 hours at 4 degrees in Zamboni's fixative, immunolabeled with antibodies against TRPV1 and SP, and imaged on a Zeiss laser scanning confocal microscope (LSM780). Neuronal TRPV1 and SP intensity were measured using ImageJ. Results: In wild-type mice, 24% of dorsal root ganglia neurons expressed TRPV1 and 26% expressed SP. In contrast, in IL5tg mice, 99% of neurons expressed TRPV1 and 97% expressed SP. TRPV1 expression in eosinophil-deficient PHIL mice was similar to wild-type control with 20% of neurons expressing TRPV1. However, SP expression in eosinophil-deficient PHIL mice was increased compared to wild-type, with 73% of neurons expressing SP. In total, 39% of TRPV1-positive neurons also expressed SP in wild-type mice whereas 97% of TRPV1-positive neurons expressed SP in IL5tg mice and 90% of TRPV1-positive neurons expressed SP in PHIL mice. Conclusions: Eosinophils increase TRPV1 and SP expression in dorsal root sensory neurons. These changes may underlie increased cough and bronchoconstriction in asthma.
Background: Toll like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded development of a mechanistic understanding of TLR7 function in nervous tissues.Methods: TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout mice, and in guinea pigs. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. Pulmonary afferent neurons in vagal ganglia were also specifically tested since respiratory viruses are a common TLR7 ligand. Guinea pig vagal afferents were labeled by intranasal instillation of wheat germ agglutinin, isolated using flow cytometry and analyzed for TLR7 by RT-PCR. The effects of influenza A infection on TLR7 expression in sensory ganglia and in spleen were also assessed.Results: In situ probes detected TLR7 in the spleens and in small support cells adjacent to sensory neurons in dorsal root and vagal ganglia in wild type mice and guinea pig, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage and satellite glial cell marker Iba1, but was absent in sensory nerves in vagal and dorsal root ganglia in both mice and guinea pigs. In contrast, a TLR7 antibody labeled small and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Wheat germ agglutinin-positive cells sorted by flow cytometry expressed both TLR7 and Iba1, indicating that TLR7-expressing support cells sort alongside neurons despite ganglia dissociation. Influenza A infection caused significant weight loss and upregulation of TLR7 in spleens, but not in vagal ganglia, in mice.Conclusion: TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7’s neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7’s role in neuronal tissues is not primarily related to antiviral immunity.
Background: Toll-like receptor 7 (TLR7) is an innate immune receptor that detects singlestranded RNA viruses and triggers an immune response. Recently, we found that airway sensory nerves express TLR7, although its role on nerves is unknown. Objective: To characterize TLR7 expression on airway sensory nerves, and to determine the effects of neuronal TLR7 stimulation. Methods: Vagal and dorsal root ganglia were isolated from female Hartley guinea pigs (~400g) and fixed in zinc formalin. Nerves were immunolabeled with antibodies against TLR7 and either neurofilament-1 (NF-H) to identify A fiber sensory nerves or transient receptor potential V1 (TRPV1) to identify C fiber sensory nerves. In separate experiments, dorsal root ganglia were isolated, cultured, and treated with the TLR7 agonist R837 (0.1-100 microM) for 16 hours. Nerve length, the number of neurites, and the number of branch points were quantified. Results: Airway sensory nerves originating in the vagal and dorsal root ganglia heterogeneously expressed TLR7. TLR7 was highly expressed on small TRPV1-expressing C fiber neurons, but not on large NF-H-positive A neurons. The TLR7 agonist R837 dose-dependently increased nerve length and branching in vitro. Conclusions: TLR7 is highly expressed by airway sensory C fibers nerves and its activation stimulates neurite growth. Neuronal TLR7 may facilitate detection of respiratory viruses and regulate anti-viral immune responses.
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